PROJECT SUMMARY The goal of this proposal is to determine how interindividual variation in heterochromatin abundance and mechanisms required for its maintenance lead to the formation and differential accumulation of spontaneous epimutations. There is exists vast amounts of heterochromatin in genomes, which varies in abundance depending on the population being studied. We hypothesize that changes to overall sequences that lead to heterochromatin formation will influence the formation of stable epimutations and therefore create novel allelic variants. Although the formation of spontaneous epimutations are documented and they are linked to a range of developmental abnormalities and diseases, the mechanisms underlying their origins is unknown. Spontaneous epimutations are rare, but this is mostly an ascertainment bias because there are have not been systematic efforts dedicated to their discovery. In this proposal we are using mutation accumulation populations from distinct genotypes including both natural variation and experimental variation as a result of induced mutations as experimental models. These experimental evolution populations make detection of spontaneous epimutations routine. At the same time, we have designed experiments to modulate the rate of epimutation formation based on varying total abundance of heterochromatin. Combined, these approaches and unique experimental populations will allow us to dissect the mechanistic origins of spontaneous epimutations, which will provide the basis for the design of drugs that can target their reversal. We expect that the proposed studies will elucidate how the mis-regulation of heterochromatin establishment or maintenance leads to spontaneous epimutations that are associated with human diseases. Heterochromatin is a critical aspect of chromosomes that is important for chromosome segregation, silencing of transposable elements and repetitive DNA sequences. Errors in proper formation of heterochromatin and its maintenance upon DNA replication are associated with a number of cellular abnormalities and human diseases. This proposal addresses a key hypothesis that posits rare epimutations in genes form as a byproduct of enzymatic activities of proteins that are required for the maintenance of heterochromatin and that they are impacted by genomic variation. These spontaneous epimutations result in a stable and inherited change in gene expression states, which is often associated with a change in DNA methylation and chromatin status. Due to the absence of changes to the DNA sequences, these spontaneous epimutations can be reversed potentially mitigating associated diseases.